Distal and proximal actions of peptide pheromone M-factor control different conjugation steps in fission yeast.

Seike T, Nakamura T, Shimoda C - PLoS ONE (2013)

Bottom Line:
Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant.When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed.Notably, M-factor-less mutant cells were also incorporated in these aggregates.

ABSTRACTMating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of "distal pheromone action", but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of "proximal pheromone action".

pone-0069491-g006: P-cells preferentially mate with wild-type M-cells.(A) Experimental design. Wild-type P-cells (PWT) were mixed with an equal number of wild-type M-cells (MWT) and M-factor-less M-cells (Mmut), marked by the ade6-M210 auxotrophic marker, in nitrogen-free medium. If the P-cell chose an ade6+ M-cell, the descendant spore clones would have no ade6-M210 allele. If the P-cell chose an ade6-M210 M-cell, the allele would be transmitted to half of the descendant spore clones. Thus, the preference of P-cells for type of mating partner could be determined by the frequency of ade6-M210 segregants. (B) Experimental results. The following strains were used: L972 (h– prototrophic), L975 (h+ prototrophic), FS120 (h–mfm1,2,3Δ ade6-M210), and FS121 (h–mfm1,2,3+ade6-M210). The mixed cells were allowed to mate, and resulting hybrid diploids were sporulated. Spores were isolated by micromanipulation and grown on nutrient medium with adenine sulfate limitation.

Mentions:
As described above, the defect in cell adhesion of M-factor-less M-cells was recovered by adding exogenous synthetic M-factor; however, exogenous M-factor did not rescue the cell fusion defect (Figure 1B), as Nielsen’s group has already observed [16]. Similarly, the sterility of map2Δ, harboring a deletion of map2+ encoding the counterpart pheromone P-factor [5], was not suppressed by the addition of synthetic P-factor (Figure 1C). Normal conjugation is attained by the down-regulation mechanism of P-factor, in which the specific protease, Sxa2, is involved [32]. Although the counterpart of Sxa2 for M-factor has not been identified, the control of M-factor concentration must also be important. We considered that the cell fusion defects may be due to fine tuning of the concentration of pheromones and the timing of pheromone addition. To verify this possibility, we conducted mixed culture experiments in which wild-type and M-factor-less M-cells, as well as wild-type P-cells, were inoculated into nitrogen-free medium (SSL−N) at a cell number ratio of 1:1:2. The M-factor-less M-type strain was genetically marked with the ade6-M210 allele. The mixed cells were cultured overnight to support mating. A pair of M- and P-cells conjugate to form a zygote, which then culminates in an ascus containing four spores. In the mixed culture, two different kinds of zygotes might be formed: one hybrid between a wild-type P-cell and a wild-type M-cell, and the other hybrid between a wild-type P-cell and an M-factor-less M-cell. In the latter type of zygote, the ascus should produce two spores harboring the ade6-M210 allele. In the case that P-cells mate equally with wild-type and mutant M-cells, the frequency of spores carrying the ade6 allele is predicted to be 25% (i.e. two of the eight spores) (Figure 6A). In the case that P-cells prefer wild-type M-cells, the frequency of mutant M-spores is expected to be less than 25% (Figure 6A).

pone-0069491-g006: P-cells preferentially mate with wild-type M-cells.(A) Experimental design. Wild-type P-cells (PWT) were mixed with an equal number of wild-type M-cells (MWT) and M-factor-less M-cells (Mmut), marked by the ade6-M210 auxotrophic marker, in nitrogen-free medium. If the P-cell chose an ade6+ M-cell, the descendant spore clones would have no ade6-M210 allele. If the P-cell chose an ade6-M210 M-cell, the allele would be transmitted to half of the descendant spore clones. Thus, the preference of P-cells for type of mating partner could be determined by the frequency of ade6-M210 segregants. (B) Experimental results. The following strains were used: L972 (h– prototrophic), L975 (h+ prototrophic), FS120 (h–mfm1,2,3Δ ade6-M210), and FS121 (h–mfm1,2,3+ade6-M210). The mixed cells were allowed to mate, and resulting hybrid diploids were sporulated. Spores were isolated by micromanipulation and grown on nutrient medium with adenine sulfate limitation.

Mentions:
As described above, the defect in cell adhesion of M-factor-less M-cells was recovered by adding exogenous synthetic M-factor; however, exogenous M-factor did not rescue the cell fusion defect (Figure 1B), as Nielsen’s group has already observed [16]. Similarly, the sterility of map2Δ, harboring a deletion of map2+ encoding the counterpart pheromone P-factor [5], was not suppressed by the addition of synthetic P-factor (Figure 1C). Normal conjugation is attained by the down-regulation mechanism of P-factor, in which the specific protease, Sxa2, is involved [32]. Although the counterpart of Sxa2 for M-factor has not been identified, the control of M-factor concentration must also be important. We considered that the cell fusion defects may be due to fine tuning of the concentration of pheromones and the timing of pheromone addition. To verify this possibility, we conducted mixed culture experiments in which wild-type and M-factor-less M-cells, as well as wild-type P-cells, were inoculated into nitrogen-free medium (SSL−N) at a cell number ratio of 1:1:2. The M-factor-less M-type strain was genetically marked with the ade6-M210 allele. The mixed cells were cultured overnight to support mating. A pair of M- and P-cells conjugate to form a zygote, which then culminates in an ascus containing four spores. In the mixed culture, two different kinds of zygotes might be formed: one hybrid between a wild-type P-cell and a wild-type M-cell, and the other hybrid between a wild-type P-cell and an M-factor-less M-cell. In the latter type of zygote, the ascus should produce two spores harboring the ade6-M210 allele. In the case that P-cells mate equally with wild-type and mutant M-cells, the frequency of spores carrying the ade6 allele is predicted to be 25% (i.e. two of the eight spores) (Figure 6A). In the case that P-cells prefer wild-type M-cells, the frequency of mutant M-spores is expected to be less than 25% (Figure 6A).

Bottom Line:
Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant.When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed.Notably, M-factor-less mutant cells were also incorporated in these aggregates.

ABSTRACTMating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of "distal pheromone action", but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of "proximal pheromone action".